The present invention relates generally to receptors implicated in neurotransmitter release, and particularly to a novel G-protein-coupled receptor which binds xcex1-latrotoxin (xcex1-LTx) independently of calcium (Ca2+) presence and is thus a regulator of neurotransmitter release which mediates the calcium-independent stimulation of neurotransmitter release by xcex1-latrotoxin.
xcex1-Latrotoxin, a vertebrate neurotoxin of Black Widow Spider venom, is a strong stimulator of spontaneous neurotransmitter release from the nerve terminal (Clark et al.,1970, Nature, 225 703-705). In physiological solutions, xcex1-latrotoxin-evoked neurotransmitter release occurs by synaptic vesicle exocytosis and is accompanied by presynaptic membrane polarization and the influx of calcium ions through the channels induced by the toxin and through presynaptic calcium channels. However, an unusual characteristic of xcex1-latrotoxin""s stimulatory action is that it does not require extracellular Ca2+, provided Mg2+ is present in the extracellular solution and can occur even without a noticeable increase in intracellular Ca2+ concentration. Stimulation of neurotransmitter release by xcex1-latrotoxin requires binding to its high-affinity membranae receptors. An immunofluorescence study of the neuromuscular junction indicates that the xcex1-latrotoxin receptors may not be restricted to the areas of synaptic contacts. Two types of receptors, differing in their calcium requirement of xcex1-latrotoxin-binding have been described. The calcium-dependent receptor has been identified as neurexin Ixcex1, a member of a large family of multiply spliced neuron-specific proteins, the neurexins. In contrast, brain glycoprotein of the Mr 120,000 which does not belong to the neurexin family recently has been purified and proposed as a calcium-independent receptor for, and a likely mediator of, the calcium-independent stimulation of neurosecretion by xcex1-latrotoxin (Krasnoperov et al., (1996), Biochem. Biophys. Res. Commun., 227:868-875 and Davletov et al., (1996) J. Biol Chem. 271, 23239-23245).
xcex1-Latrotoxin receptors have been identified biochemically, using an iodinated radioactive derivative of the toxin by Tzeng and Siekevitz, J. Neurochem. (1979) 33, 263-274; Meldolesi, J. Neurochem. (1982) 38, 1559-1569; Scheer and Meldolesi, EMBO J. (1985) 4, 323-327. These receptors were of low abundance (about 300 fmol/mg of membrane protein), and their affinity to xcex1-latrotoxin was high (Kd in the range of 10xe2x88x929-10xe2x88x9210 M). Previously, the purification of a high-affinity xcex1-latrotoxin-binding protein was identified as neurexin Ixcex1, a member of a large family of multiply spliced neuron-specific proteins, the neurexins was reported in Petrenko, FEBS Lett. (1993) 325, 81-85; Petrenko et al., (1990) EMBO J. 9, 2023-2027; and Ushkaryov et al., Science (1992) 257, 50-56. The structural features and developmental profile of neurexins suggest that they perform a function in cell adhesion or recognition important in axonogenesis, see, Ushkaryov et al., Science (1992) 257, 50-56; and Puschel and Betz, J. Neurosci. (1995) 15, 2849-2856. A hypothesis has been formulated that these highly polymorphic cell surface membrane proteins may define the specificity of synaptic connections in the brain Ushkaryov et al., Science (1992) 257, 50-56.
xcex1-Latrotoxin-binding properties of purified and recombinant neurexin-Ixcex1 are very similar to the binding properties of the membrane-bound xcex1-latrotoxin receptors with one significant exception: neurexin Ixcex1 binds xcex1-latrotoxin only in the presence of calcium, whereas the brain membranes bind the toxin even in the presence of EDTA. Therefore, neurexin Ixcex1 may be important in calcium-dependent effects of xcex1-latrotoxin, such as degeneration of nerve terminals, but not in the stimulation of neurosecretion in calcium-independent environment. Since purified xcex1-latrotoxin can form cation channels in artificial lipid bilayers, neurexin Ixcex1 may facilitate its insertion into the cellular membrane by binding with xcex1-latrotoxin, resulting in calcium fluxes through the formed cation channels. However, in the absence of calcium, this mechanism would not explain robust stimulation of spontaneous neurotransmitter release by this toxin.
In view of the above, a need therefore exists to elucidate the calcium-independent activation of xcex1-latrotoxin, and to thereby further understand its activity, and possibly devise strategies for intervention that may benefit neuronal activity. It is accordingly toward the fulfillment of these needs that the present invention is directed.
In one aspect, the present invention concerns the identification of a second xcex1-latrotoxin receptor which binds xcex1-latrotoxin both in the presence or absence of calcium and is pharmacologically and structurally distinct from neurexin Ixcex1, an earlier described receptor of xcex1-latrotoxin. This receptor, designated CIRL (calcium-independent receptor of xcex1-latrotoxin) belongs to the secretin receptor subfamily of G-protein coupled receptors, and, together with xcex1-latrotoxin, interacts with syntaxin, a component of the neuronal exocytotic machinery. CIRL, as a neuronal signaling receptor, is thus critically important for the calcium-independent stimulation of neurotransmitter release by xcex1-latrotoxin. A further aspect of the present invention involves the isolation, purification and characterization of the calcium-independent receptor of xcex1-latrotoxin (CIRL).
In its broadest aspect, the present invention extends to a novel neuronal receptor which is a regulator of neurotransmitter release, and thus mediates xcex1-latrotoxin (xcex1-LTx) toxicity both in the presence or absence of calcium.
In a specific example, the calcium-independent receptor of xcex1-latrotoxin (CIRL) has been identified as a G-protein-coupled receptor which contains a subunit of an apparent Mr 120,000, as determined by SDS-PAGE analysis.
In a still further aspect, the present invention extends to methods of utilizing CIRL. This receptor can be expressed and used to screen libraries of agents, or mixtures of natural origin (e.g., brain homogenates, detergent extracts, cell conditioned media or extracts, etc.) for ligands thereof which can then be utilized in various therapeutic methods.
Still further, since this receptor is enriched in the striatum of the mammalian brain, antibodies or nucleic acid probes thereto can be prepared which can then be utilized in diagnostic methods for screening for the presence of various neurological diseases characterized by the changes in receptor expression, or mutations thereto, or the presence of excess receptors. These diseases include, but are not limited to, neurological diseases such as schizophrenia, Alzheimer""s disease, epilepsy, stress disorder, Huntington""s disease, Parkinson""s disease, as well as peripheral neuromuscular diseases such as myasthenia gravis.
In a particular embodiment, the present invention relates to all members of the herein disclosed family of calcium-independent receptors of xcex1-latrotoxin, and to genetically engineered cells which express such receptors.
The present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a calcium-independent receptor of xcex1-latrotoxin (CIRL); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the calcium-independent receptor of xcex1-latrotoxin (CIRL), having a nucleotide sequence or is complementary to a DNA sequence shown in FIG. 2B (SEQ ID NO:2). In a still further embodiment, the gene constructs of this invention can be utilized in gene therapy in individuals wherein the lack of, or changes in, or modifications to, this receptor causes deficits in neurotransmission.
The human and murine DNA sequences of the calcium-independent receptor of xcex1-latrotoxin (CIRL) of the present invention or portions thereof, may be prepared as probes to screen for ligands, complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the calcium-independent receptor of xcex1-latrotoxin (CIRL). For example, the probes may be prepared with a variety of known vectors, such as the phage xcex vector. The present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences set forth in FIG. 2B (SEQ ID NO:2, respectively). Correspondingly, the preparation of antisense RNA and ribozymes are included herein.
The present invention also includes calcium-independent receptor of xcex1-latrotoxin (CIRL) proteins having the activities noted herein, and that display the amino acid sequences set forth and described above and selected from SEQ ID NO:1, and subunits thereof.
In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to host cells transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present calcium-independent receptor of xcex1-latrotoxin (CIRL)(s), and more particularly, the complete DNA sequence determined from the sequences set forth above and in SEQ ID NO:2.
According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active animal or human calcium-independent xcex1-latrotoxin receptor (CIRL). The transgenic animals can also include a xe2x80x9cknock-outxe2x80x9d animal as a model of the various diseases.
The concept of the calcium-independent receptor of xcex1-latrotoxin (CIRL) contemplates that specific receptors exist for correspondingly specific ligands, such as xcex1-latrotoxin and the like, as described earlier. Accordingly, the exact structure of each calcium-independent receptor of xcex1-latrotoxin (CIRL) will understandably vary so as to achieve this ligand and activity specificity. It is this specificity and the direct involvement of the calcium-independent receptor of xcex1-latrotoxin (CIRL) in the chain of events leading to release of neurotransmittors, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.
The present invention naturally contemplates several means for preparation of the calcium-independent receptor of xcex1-latrotoxin (CIRL), including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope. The isolation of the CDNA and amino acid sequences disclosed herein facilitates the reproduction of the calcium-independent receptor of xcex1-latrotoxin (CIRL) by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
The invention includes an assay system for screening of potential drugs effective to modulate activities of target mammalian cells by interrupting or potentiating the calcium-independent receptor of xcex1-latrotoxin (CIRL). In one instance, the test drug could be administered to a cellular sample with the ligand that activates the calcium-independent receptor of xcex1-latrotoxin (CIRL), or an extract containing the activated calcium-independent receptor of xcex1-latrotoxin (CIRL), to determine its effect upon the binding activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) to any chemical sample (including DNA), or to the test drug, by comparison with a control.
The assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the calcium-independent receptor of xcex1-latrotoxin (CIRL) and/or calcium-independent receptor of xcex1-latrotoxin (CIRL) factors or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating calcium-independent receptor of xcex1-latrotoxin (CIRL) activity. Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity. For example, such drugs might be used to modulate and/or reverse the degeneration of nerve terminals, to modulate synaptic transmission, or to treat other pathologies, as for example, in making calcium-independent receptor of xcex1-latrotoxin (CIRL) more resistant to xcex1-latrotoxin.
In yet a further embodiment, the invention contemplates antagonists of the activity of a calcium-independent receptor of xcex1-latrotoxin (CIRL). In particular, an agent or molecule that inhibits calcium-independent receptor of xcex1-latrotoxin (CIRL) is an embodiment of the present invention. In a specific embodiment, the antagonist can be a peptide having the sequence of a portion of an calcium-independent receptor of xcex1-latrotoxin (CIRL) domain.
One of the characteristics of the present calcium-independent receptor of xcex1-latrotoxin (CIRL) is that it is a G-coupled-protein having a subunit of apparent Mr 120,000 by SDS-PAGE Analysis.
The diagnostic utility of the present invention extends to the use of the present calcium-independent receptor of xcex1-latrotoxin (CIRL) in assays to screen for calcium-independent receptor of xcex1-latrotoxin (CIRL).
The present invention likewise extends to the development of antibodies against the calcium-independent receptor of xcex1-latrotoxin (CIRL)(s), including naturally raised and recombinantly prepared antibodies. For example, the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the calcium-independent receptor of xcex1-latrotoxin (CIRL)(s). Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating calcium-independent receptor of xcex1-latrotoxin (CIRL) activity.
In particular, antibodies against specifically phosphorylated factors can be selected and are included within the scope of the present invention for their particular ability in following activated protein. Thus, activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or of the specific polypeptides believed to be causally connected thereto may therefore be followed directly by the assay techniques discussed later on, through the use of an appropriately labeled quantity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or antibodies or analogs thereof.
Thus, the calcium-independent receptor of xcex1-latrotoxin (CIRL), their analogs and/or analogs, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the calcium-independent receptor of xcex1-latrotoxin (CIRL) that has been labeled by either radioactive addition, or radioiodination.
In an immunoassay, a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized calorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the calcium-independent receptor of xcex1-latrotoxin (CIRL), or to identify drugs or other agents that may mimic or block its activity. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the calcium-independent receptor of xcex1-latrotoxin (CIRL), their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL)(s), genetically engineered cells which express or secrete CIRL, its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the binding activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or its subunits, and comprises administering an agent capable of modulating the production and/or activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host. For example, drugs or other binding partners to the calcium-independent receptor of xcex1-latrotoxin (CIRL) or proteins may be administered to inhibit or potentiate calcium-independent receptor of xcex1-latrotoxin (CIRL) activity, as in the potentiation of calcium-independent receptor of xcex1-latrotoxin (CIRL) in therapy. Also, the blockade of the action of specific phosphatases in the dephosphorylation of activated (phosphorylated) calcium-independent receptor of xcex1-latrotoxin (CIRL) or proteins presents a method for potentiating the activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or protein that would concomitantly potentiate therapies based on calcium-independent receptor of xcex1-latrotoxin (CIRL)/protein activation.
More specifically, the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of activation of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention. For example, drugs or other binding partners to the calcium-independent receptor of xcex1-latrotoxin (CIRL) or proteins, as represented by SEQ ID NO:1, may be administered to inhibit or potentiate calcium-independent receptor of xcex1-latrotoxin (CIRL) activity as in the potentiation of calcium-independent receptor of xcex1-latrotoxin (CIRL) in therapy.
In particular, the proteins of calcium-independent receptor of xcex1-latrotoxin (CIRL) whose sequences are presented in SEQ ID NO:1 herein, their antibodies, agonists, antagonists, active fragments thereof, or expressing cells thereof, could be prepared in pharmaceutical formulations for administration in instances wherein calcium-independent receptor of xcex1-latrotoxin (CIRL) therapy is appropriate, such as to modulate and/or prevent nerve degeneration, or to modulate neurotransmitter release.
Accordingly, it is a principal object of the present invention to provide a calcium-independent receptor of xcex1-latrotoxin (CIRL) and its subunits in purified form that exhibits certain characteristics and activities associated with calcium-independent receptor of xcex1-latrotoxin (CIRL) activity.
It is a further object of the present invention to provide antibodies to the calcium-independent receptor of xcex1-latrotoxin (CIRL) and its subunits, and methods for their preparation, including recombinant means.
It is a further object of the present invention to provide a method for detecting the presence of the calcium-independent receptor of xcex1-latrotoxin (CIRL) and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.
It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or combating the adverse effects of the calcium-independent receptor of xcex1-latrotoxin (CIRL) and/or its subunits in mammals.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or subunits thereof, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the calcium-independent receptor of xcex1-latrotoxin (CIRL) or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the calcium-independent receptor of xcex1-latrotoxin (CIRL), its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of the calcium-independent receptor of xcex1-latrotoxin.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.